Tritc conjugated phalloidin

Following cell adhesion on Ti discs for 20 min, the actin cytoskeletal organization was observed by staining cells with TRITC-conjugated Phalloidin^{26 (link)}. Cells were briefly washed twice with PBS and fixed for 30 min with 4% formaldehyde before being permeabilized with 0.5% (v/v) Triton-X 100 for 20 min at room temperature. After that, cells were incubated in a dark moist chamber for 40 min with TRITC-conjugated Phalloidin (Sigma, St. Louis, MO, USA) before being washed twice with PBS and viewed under a fluorescent microscope (EVOS M5000 Imaging System, Thermo Fisher Scientific, USA).
For confocal imaging, the hFOB 1.19 cells were stained with TRITC-conjugated Phalloidin and then washed with PBS, cells were counterstained with DAPI (Sigma, St. Louis, MO, USA) for 5 min. The fine details on the organization of the actin cytoskeleton were observed by confocal microscope ZEISS LSM 900 with Airyscan 2 (Carl-Zeiss, Germany).

Cells were plated in confocal dishes for two days followed by fixation in 4% paraformaldehyde at room temperature for 20 min. After permeabilization with 0.1% Triton X-100 for 5 min, cells were incubated in 1%BSA in PBS for 1 h at room temperature. Cells were then incubated overnight with primary antibodies against ZO-1 (1:500, Proteintech) followed by incubation with goat anti-Rabbit Secondary Antibody, Alexa Fluor 488 (1: 5000, Invitrogen), and TRITC-conjugated Phalloidin (1:1000, Sigma-Aldrich). Staining of focal adhesion was conducted with the Actin Cytoskeleton and Focal Adhesion Staining Kit (Merck Millipore). For antigen staining, cells were incubated with anti-vinculin primary antibody (1:200, Merck Millipore) overnight at 4 °C, followed by incubation with a secondary FITC-conjugated antibody (1:5000, Proteintech) and TRITC-conjugated Phalloidin (1:1000, Sigma-Aldrich) for 1 h, respectively. Nuclei counterstaining was performed by incubating cells with DAPI for 5 min at room temperature. The quantification of focal adhesion (FA) number, actin and ZO-1 intensity were determined using ImageJ software (Version 1.8.0). Original data were displayed in supplementary data.

Leaching solutions were used to investigate the effect of scaffolds on cell’s cytoskeleton arrangement. For leaching solutions preparation, the three sterilized materials were soaked in complete growth media (DMEM containing 10% fetal bovine serum and 1% Penicillin/Streptomycin) at 37°C sterile incubator for 48 h, respectively. After then, the supernatant was collected as a leaching solution and stored at 4 °C for subsequent cell culture. After L929 cells were seeded in a 24-well culture plate, the leaching solutions were added as a culture medium. After 3 days of culture, cells were washed 2 times with PBS at ambient temperature and then fixed in 4% paraformaldehyde for 40 min. Subsequently, cells were stained with TRITC-conjugated phalloidin (1:200, sigma) in dark for 40 min. After being washed gently, cells were stained with DAPI (1:200, sigma) for 5 min and washed with PBS again. Finally, the cell’s cytoskeleton was visualized by confocal laser scanning microscopy (Leicasp5, Germany) and fluorescence microscope (80i, Japan).